Process for the enzymatic production of cyclic diguanosine monophosphate employing a diguanylate cyclase comprising a mutated rxxd motif

Abstract

A process is disclosed for the production of cyclic di-guanosine monophosphate (c-di-GMP) without the use of protecting groups by means of an enzymatic synthesis. The process comprises the coupling of two guanosine triphosphate (GTP) molecules so as to form a c-di-GMP molecule. This is done under the influence of a mutant diguanylate cyclase (DGC) comprising the amino acid sequence V153M154G155G156. It has been found that the DGC is obtainable from inclusion bodies, and therewith can be made available in amounts sufficient to improve the c-di-GMP synthesis. Particularly, the latter synthesis can be conducted in a one-pot method starting from commercially available bulk chemicals and allows upscaling to a commercial production scale.

1 . A mutant diguanylate cyclase (DGC) comprising a modified RXXD motif, wherein the DGC is provided in the form of inclusion bodies. 
     
     
         2 . A mutant DGC according to  claim 1 , comprising a modified RXXD motif at amino acids 153-156, selected from the group consisting of GMGG (SEQ ID NO.: 2), VMGG (SEQ ID NO.: 1), GGVA (SEQ ID NO.: 4), GRDC (SEQ ID NO.: 5), GVGD (SEQ ID NO.: 6), MEGD (SEQ ID NO.: 7), GGNH (SEQ ID NO.: 8), RESE (SEQ ID NO.: 9), RNRD (SEQ ID NO.: 10), RVDS (SEQ ID NO.: 11), RAGG (SEQ ID NO.: 12), and RGQD (SEQ ID NO.: 13). 
     
     
         3 . A mutant DGC according to  claim 2 , having the  C. crescentus  DgcA (CC3285) amino acid sequence V153M154G155G156. 
     
     
         4 . A method for the manufacture of recombinant DGC according to  claim 1 , comprising the over-expression of a suitable DGC gene in a suitable host cell, and harvesting the DGC from inclusion bodies thereby obtained. 
     
     
         5 . A process for the production, by enzymatic synthesis, of cyclic di-guanosine monophosphate (c-di-GMP) comprising the coupling of two guanosine triphosphate (GTP) molecules so as to form a c-di-GMP molecule, under the influence of a mutant diguanylate cyclase (DGC) comprising a modified RXXD motif, conducted in a one-pot reaction wherein GTP is formed by the conversion of guanosine monophosphate (GMP), comprising the addition, to a suitable reaction medium, of (a) GMP, (b) a phosphate anhydride donor, (c) a guanylate kinase (GmpK), (d) a nucleoside-diphosphate kinase (NdK), and (e) the DGC, mixing, and incubating the reaction mixture, so as to form c-di-GMP. 
     
     
         6 . A process according to  claim 5 , wherein the DGC is refolded DGC obtainable from inclusion bodies. 
     
     
         7 . A process according to  claim 5 , wherein the DGC is used in an amount of 0.1 μM-10 μM. 
     
     
         8 . A process according to  claim 7 , wherein the DGC is used in an amount of 1-2 μM. 
     
     
         9 . A process for the production, by enzymatic synthesis, of cyclic di-guanosine monophosphate (c-di-GMP) comprising the coupling of two guanosine triphosphate (GTP) molecules so as to form a c-di-GMP molecule, under the influence of a mutant diguanylate cyclase (DGC) comprising a modified RXXD motif, wherein the DGC is provided in the form of inclusion bodies. 
     
     
         10 . A process according to  claim 5 , wherein the DGC is supplemented during the synthesis. 
     
     
         11 . A batch of at least 10 g of c-di-GMP of over 99% purity, obtainable by a process according to  claim 5 . 
     
     
         12 . A batch according to  claim 11 , having a size of at least 100 g. 
     
     
         13 . The process of  claim 6 , wherein the mutant DGC comprises a modified RXXD motif at amino acids 153-156, selected from the group consisting of GMGG (SEQ ID NO.: 2), VMGG (SEQ ID NO.: 1), GGVA (SEQ ID NO.: 4), GRDC (SEQ ID NO.: 5), GVGD (SEQ ID NO.: 6), MEGD (SEQ ID NO.: 7), GGNH (SEQ ID NO.: 8), RESE (SEQ ID NO.: 9), RNRD (SEQ ID NO.10), RVDS (SEQ ID NO.: 11), RAGG (SEQ ID NO.: 12), and RGQD (SEQ ID NO.: 13). 
     
     
         14 . The process of  claim 6 , wherein the mutant DGC has the  C. crescentus  DgcA (CC3285) amino acid sequence V153M154G155G156. 
     
     
         15 . The process of  claim 9 , wherein the mutant DGC comprises a modified RXXD motif at amino acids 153-156, selected from the group consisting of GMGG (SEQ ID NO.: 2), VMGG (SEQ ID NO.: 1), GGVA (SEQ ID NO.: 4), GRDC (SEQ ID NO.: 5), GVGD (SEQ ID NO.: 6), MEGD (SEQ ID NO.: 7), GGNH (SEQ ID NO.: 8), RESE (SEQ ID NO.: 9), RNRD (SEQ ID NO.10), RVDS (SEQ ID NO.: 11), RAGG (SEQ ID NO.: 12), and RGQD (SEQ ID NO.: 13). 
     
     
         16 . The process of  claim 9 , wherein the mutant DGC has the  C. crescentus  DgcA (CC3285) amino acid sequence V153M154G155G156. 
     
     
         17 . A method for the manufacture of recombinant DGC according to  claim 2 , comprising the over-expression of a suitable DGC gene in a suitable host cell, and harvesting the DGC from inclusion bodies thereby obtained. 
     
     
         18 . A method for the manufacture of recombinant DGC according to  claim 3 , comprising the over-expression of a suitable DGC gene in a suitable host cell, and harvesting the DGC from inclusion bodies thereby obtained.